Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.17 (lysozyme)
21,489 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A new method has been developed which permits the rapid screening of E. coli colonies for mutants with defective enzymes of phospholipid metabolism. In this procedure, a disc of filter paper is pressed down on an agar plate containing several hundred colonies of mutagen-treated cells, after which the paper is lifted off. In the process the colonies are transferred to the paper, giving rise to a replica print of the master plate. The few cells from each colony left on the master keep growing in the original pattern. The pattern of colonies is also retained on the filter paper, even after the cells are rendered permeable with lysozyme and EDTA. Colonies treated in this manner remain absorbed to the paper, where they can convert sn-(U-14-C)glycero-3-P to phosphatidyl(U-14-C)glycerophosphate, dependent on added CDP-diglyceride. Unrelated reactions of sn-(U-14-C)glycero-3-P that may obscure the synthesis of phosphatidyl-glycerophosphate are inhibited by the addition of reagents poisoning energy generation. The radioactive phospholipid that forms around each colony on the paper is precipitated in situ with trichloroacetic acid, and unreacted sn-(U-14-C)glycero-3-P is washed away. After autoradiography, the colonies on the filter paper are stained with Coomassie blue. When the autoradiogram is superimposed on the strained paper, mutants are identified as blue colonies lacking a black halo. With this method, 20,000 colonies were screened in several days. Four mutants were identified with low levels of CDP-diglyceride:snglycero-3-P phosphatidyl transferase (EC 2.7.8.5, GLYCEROL-PHOSPHATE PHOSPHATIDYLTRANSFERASE, PHOSPHATIDYLGLYCEROPHOSPHATE SYNTHETASE) IN EXTRACTS. With a similar assay, 10,000 additional colonies were screened for mutants with altered CDP-diglyceride:L-serine O-phosphatidyltransferase (EC 2.7.8.8, phosphatidylserine synthetase), and four strains were found in which the enzyme is thermolabile. The screening technique described here is termed replica printing and should be applicable not only to studies of phospholipid metabolism but also to nucleic acid and protein synthesis.
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PMID:Isolation of Escherichia coli mutants defective in enzymes of membrane lipid synthesis. 4 56

In the studies presented the effective procedure of isolation and purification of enterobacterial common antigen from Shigella sonnei has been elaborated. The method is based on sonification of bacterial suspension in the presence of lysozyme and EDTA and subsequent extraction of the pellet with boiling water. The crude extract of common antigen was purified by fractionation with ethanol and chromatography on silica gel and Sephadex LH-20. The comparison of several extraction procedures of enterobacterial common antigen from Shigella sonnei proved that the method described above is most effective. The purified enterobacterial common antigen preparation obtained preserved full biological activity: antigenicity (precipitation and activity in enzyme-linked immunosorbent assay), immunogenicity in rabbits, ability to coat erythrocytes (passive hemagglutination) and inhibitory activity in passive hemagglutination. The pure enterobacterial common antigen was identified to 90% as a polymer of N-acetyl-D-mannosaminuronic acid and N-acetyl-D-glucosamine (2:1, molar ratio), O-acetylated and containing 3.2% fatty acids (C16:0 and C18:1, not oleic). It contains 5.3% nitrogen, less than 4% protein, less than 0.5% phosphorus and less than 1.6% neutral sugar; glycerol and RNA were not found in the preparation.
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PMID:Enterobacterial common antigen: isolation from Shigella sonnei, purification and immunochemical characterization. 10 11

In vitro and "in situ" assays have been developed to test the carbamyl phosphate synthetase (CPSase) activity of a series of pyrimidine-requiring mutants of Bacillus subtilis. The enzyme has been shown to be highly unstable, and was successfully extracted only in the presence of 10% glycerol and 1 mM dithiothreitol (Cleland's reagent). It loses activity rapidly when sonicated or when treated with lysozyme. Genetic studies, using mutants, indicate that B. subtilis may possess two CPSases. This possibility and its physiological consequences were probed enzymatically. CPSase activity has been shown to undergo inhibition by both uridine triphosphate and dihydroorotate; activation has been demonstrated in response to phosphoribosyl pyrophosphate (PRPP) and (to a lesser extent) ornithine.
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PMID:Carbamyl phosphate synthesis in Bacillus subtilis. 23 5

We describe a simple, rapid, sensitive, and highly reproducible assay for lysozyme, with use of concentrated cell suspensions of Micrococcus lysodeikticus in Tris-buffered glycerol/water (40/60 by vol), pH 7.5. Stored at -20 degrees C, the cells' susceptibility to lysozyme remains unaltered over long periods. Almost identical concentration curves were obtained with different aliquots of the same preparation during eight months. Lysozyme activity was reflected in the decrease in absorbance of the reaction mixture after incubation for 15 min at 37 degrees C. Concentrations of egg-white lysozyme as low as 0.02 mg/L can be accurately assayed.
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PMID:Improved lysozyme assay in biological fluids. 26 92

Minicells produced by Bacillus subtilis CU403 (divIVB1) are capable of mucopeptide biosynthesis as shown by the incorporation of L-alanine, D-alanine, and N-acetylglucosamine into trichloroacetic acid-precipitable material, which can be degraded to trichloroacetic acid-soluble material by lysozyme digestion. Incorporation of the precursors is sensitive to vancomycin and D-cycloserine and insensitive to chloramphenicol. Penicillin inhibits the incorporation of D- and L-alanine N-acetylglucosamine at concentrations in excess of 10 mug of penicillin per ml; however, minicells are insensitive to penicillin-induced lysis. The material synthesized in minicells from N-acetylglucosamine is not subject to turnover during a subsequent 6-h incubation period. [2-3H]glycerol is converted to a cold trichloroacetic acid-precipitable form by minicells. This synthesis is not inhibited by vancomycin, penicillin, D-cycloserine, or chloramphenicol. Fractionation of the material synthesized from glycerol into hot trichloroacetic acid-soluble material and chloroform/methanol-extractable material indicates that minicells convert glycerol into teichoic acid and lipid.
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PMID:Synthesis of cell envelope components by anucleate cells (minicells) of Bacillus subtilis. 40 71

Sugars and polyols stablize proteins against heat denaturation. Scanning calorimetry was used to obtain a quantitative estimate of the degree of stabilization. Solutions of ovalbumin, lysozyme, conalbumin, and alpha-chymotrypsinogen were heated at a constant rate, and the temperature of the maximum rate of denaturation was estimated (Tm). Addition of a sugar or polyol raised Tm. The magnitude of the stabilizing effect (delta Tm) depended on both the nature of the protein and the nature of the sugar or polyol, ranging from 18.5 degrees C for lysozyme at pH 3 in the presence of 50% (w/w) sorbitol to 0 degrees C for conalbumin at pH 7 in 50% glycerol solution. It is argued that this stablization is due to the effects of sugars and polyols on hydrophobic interactions. The strength of the hydrophobic interaction was measured in model systems in sucrose and glycerol solutions. Sucrose and glycerol strengthened the pairwise hydrophobic interaction between hydrophobic groups; however, they reduced the tendency for complete transfer of hydrophobic groups from an aqueous to a nonpolar environment. The extent of stabliziation by different sugars and polyols is explained by their different influences on the structure of water. The difference between the partial molar volume of the sugar or polyol and its van der Waals volume was used as a rough quantitative measure of the structure-making or structure-breaking effect. There was a linear relationship between this quantity and delta Tm.
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PMID:Increased thermal stability of proteins in the presence of sugars and polyols. 49 77

A new system in which the in vivo and in vitro formation of cross-links in the peptidoglycan of Bacillus megaterium can be compared directly has been developed. The method for the determination of the in vivo cross-linking consists of lysozyme digestion of acetylated [(14)C]diaminopimelic acid-labeled cells and Bio-Gel P-6 gel filtration of the digest. The elution profile indicates the cell wall synthesized in vivo consists of highly cross-linked fractions (44%), bisdisaccharide peptide(s) (38%), and disaccharide peptide(s) (18%). The in vitro system showed a high synthetic activity of cross-linked peptidoglycan. The synthesis was inhibited completely by 83.3 mug of ristocetin or vancomycin per ml or 10(-4) M p-chloromercuribenzoate and inhibited only partially by penicillins. The polymerization was stimulated by high concentrations of sucrose, glycerol, amino acids, or dimethyl sulfoxide. The formation of cross-links was inhibited 50% at 0.3 mug of dicloxacillin per ml and 90% at 0.5 mug or more. It was also stimulated by high concentrations of sucrose, glycerol, or dimethyl sulfoxide. Effective concentrations of dicloxacillin on the growth, viability, and morphology of B. megaterium were determined. Sharp inhibition of cross-linking occurred in vivo and in vitro at these effective concentrations, whereas the incorporation of [(14)C]-diaminopimelate into bacterial cells was not affected at all. Cell-bound dicloxacillin reduced severely the degree of cross-linking in the cell wall synthesized after transfer to a dicloxacillin-free medium. Cell wall synthesized in the presence of dicloxacillin showed a higher rate of turnover than did the normal cell wall. Moreover, disaccharide peptide(s) was degraded faster than was bisdisaccharide peptide(s) in dicloxacillin-treated cells. From these observations, the primary target of penicillin action in B. megaterium is discussed in relation to the inhibition of cross-linking, penicillin-binding components, and cell lysis.
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PMID:Mode of action of penicillins in vivo and in vitro in Bacillus megaterium. 82 31

It is known that two types of high-molecular-mass protease complexes are present in the cytosol of mammalian cells; a 20S latent multicatalytic proteinase named the proteasome, and a large proteolytic complex with an apparent sedimentation coefficient of 26S that catalyzes ATP-dependent breakdown of proteins conjugated with ubiquitin. In this work, we first demonstrated that a low concentration of SDS was required for activation of the latent proteasome, whereas the 26S complex degraded substrates for proteasomes in the absence of SDS. Moreover, the 26S complex was greatly stabilized in the presence of 2 mM ATP and 20% glycerol. Based on these characteristics, we next devised a novel procedure for purification of the 26S proteolytic complexes from human kidney. In this procedure, the proteolytic complexes were precipitated from cytoplasmic extracts by ultracentrifugation for 5 h at 105000 x g, and the large 26S complexes were clearly separated from the 20S proteasomes by molecular-sieve chromatography on a Biogel A-1.5 m column. The 26S enzyme was then purified to apparent homogeneity by successive chromatographies on hydroxyapatite and Q Sepharose, then by glycerol density-gradient centrifugation. Electrophoretic and immunochemical analyses showed that the purified human 26S complex consisted of multiple subunits of proteasomes with molecular masses of 21-31 kDa and 13-15 protein components ranging in molecular mass over 35-110 kDa, which were directly associated with the proteasome. The purified 26S proteolytic complex degraded 125I-labeled lysozyme-ubiquitin conjugates in an ATP-dependent manner. The 26S enzyme also showed high ATPase activity, which was copurified with the complex. Vanadate and hemin strongly inhibited not only ATP cleavage, but also ATP-dependent breakdown of ubiquitinligated proteins, suggesting that the 26S complex hydrolyzes ATP and ubiquitinated proteins by closely linked mechanisms. These findings indicate that the 26S complex consists of a proteasome with proteolytic function and multiple other components including an ATPase that regulates energy-dependent, ubiquitin-mediated protein degradation.
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PMID:Demonstration that a human 26S proteolytic complex consists of a proteasome and multiple associated protein components and hydrolyzes ATP and ubiquitin-ligated proteins by closely linked mechanisms. 131 98

We have identified and purified a protein complex from human red blood cells that activates the multicatalytic protease (MCP). The complex, which we call the regulator, sediments at 11 S and is composed of 30-kDa subunits. The regulator does not hydrolyze fluorogenic peptides, but when multicatalytic protease and regulator are combined, MCP cleaves succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin and Leu-Leu-Glu-p-nitroanilide as much as 60-fold faster. Hydrolysis of several other fluorogenic peptides is stimulated to a lesser extent, and activated MCP does not degrade ubiquitin-lysozyme conjugates, bovine serum albumin, or lysozyme. Latent and activated forms of MCP display similar sensitivity to protease inhibitors, suggesting that activation does not generate new kinds of catalytic sites. In addition, ATP suppresses peptide hydrolysis by activated and latent MCPs to the same extent. Activation involves binding of regulator to MCP, and activated MCP migrates slower on native acrylamide gels. Dissociation of the MCP regulator complex during prolonged sedimentation on glycerol gradients releases active regulator and MCP molecules capable of being reactivated. Moreover, two-dimensional electrophoresis does not reveal changes in MCP or regulator subunits following activation. Thus, activation appears to result from reversible association of regulator subunits with MCP.
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PMID:Purification of an 11 S regulator of the multicatalytic protease. 142 90

When reactions take place with one of the reactants tied to protein matrix, movements along the reaction coordinate towards the transition state can become coupled to structural fluctuations of the protein matrix. This investigation aims to test the assumptions underlying the arguments supporting such a coupling. A coupling is allowed only if the activation barrier is high and broad enough as shown to be the case for the proton catalyzed isotope exchange at Trp-63 of lysozyme. In the present investigation the activation barrier for the same reaction has been lowered radically in an effort to show that the coupling, as measured by the dependence of rate on solution viscosity, will diminish and ideally vanish, despite the unchanged effects of cosolvents on the chemical activities of all the reactants. The isotope exchange rate at the indole nitrogen of the single tryptophan residue of human serum albumin was measured with UV. This residue is rigidly held to the protein surface and the solvent access, although restricted, corresponds to a partially exposed residue. As a consequence, the isotope exchange rates and the bimolecular quenching rate of fluorescence by acrylamide, also measured, are high. The experiments were carried out at pH 5.2 where the molecule is in the N-form and the exchange is catalyzed by OH- ions. The activation energy of the hydroxyl catalyzed reaction is 22 kJ lower than for the proton catalyzed process. Under these conditions the exchange rate is viscosity independent both in the case of glycerol and in ethylene glycol.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The effect of viscosity on the accessibility of the single tryptophan in human serum albumin. 158 18


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